The long-range objective of this project, now in its 14th year, is to define the sequence of steps and mechanisms involved in the regeneration of synapses. It has focused on the neuromuscular synapse because of its convenience for experimentation. The experiments outlined in this proposal concern the mechanisms that regulate formation of the structural specializations in axon terminals and muscle fibers crucial for synaptic transmission. Most of the experiments are directed toward defining the structure, function and regulation of the protein agrin. Such studies will enable us to test the hypothesis that agrin at the neuromuscular junction mediates the nerve-induced aggregation of acetylcholine receptors, acetylcholinesterase and other molecules that compose the synaptic specializations on muscle fibers. Some of the studies are also directed toward determining the molecular basis of the muscle-induced formation of synaptic specializations in axon terminals. The specific aims are: 1. To determine the cellular source of agrin active at the neuromuscular synapse. 2. To identify and characterize function domains in agrin. 3. To study the regulation of agrin during development and regeneration. 4. To identify and characterize molecules that induce the formation of presynaptic apparatus in regenerating axon terminals. These experiments will involve light and electron microscopy, immunocytochemistry and molecular genetics and will be conducted on a variety of nerve-muscle preparations. Studies such as these are requisite for understanding the cellular and molecular basis of neuromuscular disease and for devising ways to enhance restoration of neuromuscular function after trauma. Because synapses in the brain have pre- and postsynaptic specializations similar to those at the neuromuscular synapse, these studies may also provide insight as to the mechanisms involved in CNS synapse formation.